2,4,6-trimethylbenzene-1,3-diamine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 2021; finalised 12.01.2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

According to the guidance document No 263 on INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) for serious eye damage and eye irritation non OECD adopted in vitro test methods can be conducted for an weight of evidence analysis. If no decision regarding classification and labelling can be drawn according to the performed guideline tests, other in vitro test methodes for serious eye damage & eye irritation can be conducted even. As mentioned in the IATA 263 these in vitro test methods not even need to be adopted by the OECD.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Reference 2

Reference Type:

publication

Title:

Impedance Spectroscopy for the Non Destructive Evaluation of In Vitro Epidermal Models

Author:

Groeber, F et al.,

Year:

2015

Bibliographic source:

Groeber, F Engelhardt, L Egger, S Werthmann, H Monaghan M Walles H Hansmann, J (2015). Impedance Spectroscopy for the Non Destructive Evaluation of In Vitro Epidermal Models Pharmaceutical Research, 32 (5), 1845-1854. doi 10 1007 /s 11095 014 1580 3

Reference 3

Reference Type:

publication

Title:

Replacing the Draize eye test Impedance spectroscopy as a 3R method to discriminate between all GHS categories for eye irritation

Author:

Lotz, C et al.,

Year:

2018

Bibliographic source:

Lotz, C Kiesewetter, L Schmid, F F et al., Replacing the Draize eye test Impedance spectroscopy as a 3 R method to discriminate between all GHS categories for eye irritation. Sci Rep 8, 15049 (2018) https :://doi org/ 10 1038 /s 41598 018 33118 2

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted June 14, 2019

Deviations:

yes

Remarks:

used cornea model: inhouse developed reconstructed cornea model ( RCE)

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,4,6-trimethylbenzene-1,3-diamine

EC Number:

221-456-9

EC Name:

2,4,6-trimethylbenzene-1,3-diamine

Cas Number:

3102-70-3

Molecular formula:

C9H14N2

IUPAC Name:

2,4,6-trimethylbenzene-1,3-diamine

Details on test material:

2,4,6-trimethylbenzene-1,3-diamine from Evonik, Batch: 10330010

Specific details on test material used for the study:

solid

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

The in vitro eye irritation test ( is based on the protocol of the OECD test guideline 492 with the Fraunhofer-in house developed reconstructed cornea model (RCE).

All experiments were conducted in compliance with the rules for investigation on human subjects, as defined in the Declaration of Helsinki. Informed consent was obtained from all subjects prior to the study. Human corneal cells were isolated from spare limbal rings and from corneas that failed the quality criteria for clinical use in accordance and with the approval of the local ethics committee (Ethik-Kommission der Universität Würzburg, approval number 182/10) and the informed consent of the patients for the study participation. Corneal biopsies were provided by the eye clinic of the Universitätsklinikum Würzburg (Würzburg, Germany). No tissues were procured from prisoners.
Corneal tissues were transferred and washed in a petri dish with phosphate-buffered saline. Subsequently, the cornea was cut into horizontal stripes of about 2–3 mm and put in a petri dish with dispase [2 U/ml] (Thermo Fisher Scientific, Waltham, MA USA)) for 18 hours at 4 °C. The epithelium was stripped off the central cornea to the limbus with forceps and collected in a new petri dish with fresh phosphate-buffered saline. The epithelial sheets were centrifuged and reduced to small pieces by pipetting for cell seeding. The corneal epithelium was cultured in corneal epithelial cell medium (LGC Standards, Wesel, Germany). The RCE was generated from human corneal epithelial cells. The cells were seeded as described above with a cell density of 2.5 × 105 cells/cm2 and cultured for 11 days.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 mg solid
50 µl liquid (positive and negative control)

Duration of treatment / exposure:

liquids: 30 ± 2 min
solids: 6 + 0.25 h

Observation period (in vivo):

up to 11 days

Duration of post- treatment incubation (in vitro):

liquids: 120 ± 15 min
solids: 18 + 0.25 h

Number of animals or in vitro replicates:

2x RCE were used for each assays and treatment group (test item, positive and negative control), 12x in vitro corneas in total.

Details on study design:

The in vitro eye irritation test ( is based on the protocol of the OECD test guideline 492 with the in house developed reconstructed cornea model (RCE). The EIT is complemented by the integration of impedance spectroscopy for long term observation of the applied tissue models. The RCE are pre-wet with 20 μl of phosphate buffered saline for 30 min. Each assay and each treatment (test item, positive & neg. control) were conducted in duplicate. Therefore,12 RCE were used in total. Duplicates of cornea epithelia models, are exposed for 30 min ± 2 min (liquids) or 6 h ±15 min (solids) via topical application of the test material with 50 μl (liquids) or 50 mg (solids) at 37 C, 5% CO2 and 95% humidity in an incubator. Tissue models treated with water (CAS RN 7732 18 5) are used as negative control or 10% Benzalkonium chloride (CAS RN 63449 41 2 as positive control. After exposure the test substances are rinsed through three washing steps at room temperature (RT). An additional post-soak of 12± 2 min (liquids) or 25± 2 min (solids) ensures the removal of excess substance by immersing the models in 5 ml Epilife Medium (Life Technologies, MEPI 500 CA) at RT. Subsequently, viability is measured after post incubation of 120 ± 15 min (liquids) or 18 ±0.25 h (solids) at 37C, 5% CO2 and 95% humidity. Transepithelial electrical resistance at 1000 Hz (TEER 1000 Hz) measurements are performed with a customized device. Barrier integrity of tissue models are evaluated via impedance spectroscopy prior to substance application and after the post incubation and is repeated on day 1, 3, 7 and 11 after test material application. In this study the test protocol for solids was applied. Acceptability range for negative control before treatment 4000 >NC <600 Ohm/cm².

The following destructive endpoint measurements are performed:
- Tissue viability of two RCE is quantified by MTT reduction after post incubtaion
The following non destructive measurements are performed for each test material:
- Barrier integrity of two RCE is quantified by impedance spectroscopy before application and after application at day 0 1 3 7 and 11

VIABILITY measurments (MTT):
The assessment of model viability was conducted based on the protocol as described in OECD test guideline 492. Briefly, reconstructed corneal epithelial models were placed in 200 μl MTT solution 1 mg/mL in PBS)(Sigma Aldrich, M 2128 1 G) for 3 h in a humidified atmosphere with 37C and 5% CO2. The MTT reduction was then quantified by extracting the precipitated blue formazan salt with 2 ml 2-propanol and measuring the optical density of the extract at a wavelength at 570 nm using a spectrophotometer (InFinite M Nano, Tecan). After correcting thedata using 2-propanol as blank the relative tissue viability was calculated for each tissue by normalizing the corrected optical density values to the negative control which was set to 100. Acceptability range for negative control 2.5> OD> 0 8.

IMPEDACE SPECTROSCOPY (TEER 1000 Hz):
Impedance spectroscopy allows for a noninvasive evaluation of a tissue model of interest by regarding the tissue’s electrical characteristics Employing a measuring system developed by the Fraunhofer one can identify biologically relevant electrical parameters from impedance spectra in a frequency range from 1 Hz to 100 kHz. Experimental studies revealed a strong correlation between these electrical parameters and the state of the tissue models (Groeber et al ., 2015) . Compared to conventional methods, impedance spectroscopy shows a higher sensitivity in the detection of perturbations within the non viable cell layers. Therefore, an electrode is installed on top and a second one underneath a model and a low current is introduced. Since the walls of the carrier inserts are nonconductive, the complex alternating current resistance (impedance) can be yielded from the sloping voltage at a certain frequency point It could be asserted that the impedance at 1000 Hz multiplied by the culture area of the carrier inserts suits best answering the initial issues of the present study. This transepithelial electrical resistance (TEER) at 1000 Hz (TEER 1000 Hz) can be measured repeatedly during culture, which allows that the exact same models can be compared as this measuring technique works noninvasive. The results are presented in relative values normalized to the negative control. Barrier integrity of tissue models are evaluated via impedance spectroscopy prior to substance application and after the post incubation and is repeated on day 1, 3, 7 and 11 after test material application.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

TEER value (Transepithelial Electrical Resistance) 

Remarks:

at 1000 Hz

Run / experiment:

impedance spectroscopy at 1000 Hz day 7d

Value:

14

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

< 50% indicating a Category 1 substance by TEER measurement

Results 2

Irritation parameter:

TEER value (Transepithelial Electrical Resistance) 

Remarks:

at 1000 Hz

Run / experiment:

impedance spectroscopy at 1000 Hz at day 1

Value:

50

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

50% < 60% cut-off

Results 3

Irritation parameter:

mean percent tissue viability 

Remarks:

% of negative control

Run / experiment:

MTT

Value:

1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

category 1 or 2 by viability

Other effects / acceptance of results:

ACCEPTANCE of RESULTS:
- Acceptance criteria met for viability assay: yes
- Acceptance criteria met for TEER measurments: yes
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Any other information on results incl. tables

	Individual values of in vitro Eye Irritation Viability
Treatment	Replicate	individual raw OD540 nm	individual OD540 nm blank corrected	mean OD540 nm (SD)	OD540 nm of 2 tissues (SD)	individual Viability % (SD)	mean Viability % (SD)
Negative control	tissue 1	1.2433	1.2017	1.2473	1.2793	97.50	100.00
Water		1.3344	1.2928	(0.06)	(0.05)		(3.54)
	tissue 2	1.3226	1.281	1.3113		102.50
		1.3831	1.3415	(0.04)
Positive control	tissue 1	0.0456	0.004	0.0042	0.3244	0.32	0.36
10 % benzalkonium chloride		0.0459	0.0043	(0.0002)	(0.0006)		(0.05)
	tissue 2	0.0464	0.0048	0.0050		0.39
		0.0468	0.0052	(0.0003)
Test item	tissue 1	0.0549	0.0133	0.0122	0,00885	0.95	0.69
		0.0527	0.0111	(0.002)	(0.0047)		(0.37)
	tissue 2	0.0475	0.0059	0.0055		0.43
		0.0467	0.0051	(0.0006)
Blank	tissue 1	0.0408		0.0416
		0.0424
OD:	Optical density
SD:	Standard deviation

 

 

Table 2:

Individual values of in vitro Eye Irritation Transepithelia electrical resistance at 1000 Hz
		individual raw TEER1000Hz data black corrected			individual normalized TEER %
Treatment	Timepoint			TEER1000Hz of 2 tissues			TEER % of 2 tissues
		tissue 1	tissue 2		tissue 1	tissue 2	mean	SD
Negative control	Day 0 before	970.0	1126.5	1048.2	92.54	107.46	100.00	10.6
Water	Day 0 after	944.2	1078.3	1011.2	93.37	106.63	100.00	9.4
	Day 1	920.7	934.5	927.6	99.26	100.74	100.00	1.1
	Day 3	990.4	1179.9	1085.1	91.27	108.73	100.00	12.3
	Day 7	1719.1	1639.3	1679.2	102.38	97.62	100.00	3.4
	Day 11	2813.8	N/A	2813.8	100.00	N/A	100.00	N/A
Positive control	Day 0 before	1611.5	1625.4	1618.4	153.73	155.06	154.40	0.9
10 % benzalkonium chloride	Day 0 after	22.3	7.8	15.1	2.21	0.77	1.50	1
	Day 1	15.9	11.1	13.5	1.71	1.20	1.50	0.4
	Day 3	-11.9	-1.1	-6.5	1.10	-0.10	-0.60	0.7
	Day 7	3.6	4.6	4.1	0.21	0.28	0.20	0
	Day 11	43.8	N/A	43.8	1.56	N/A	1.60	N/A
Test Item	Day 0 before	1182.3	1087.9	1135.1	112.76	103.78	108.30	6.4
	Day 0 after	384.1	623.9	504	37.98	61.70	49.80	16.8
	Day 1	301.9	525.2	413.6	32.55	56.62	44.60	17
	Day 3	265.7	338.8	302.2	24.28	31.22	27.90	4.8
	Day 7	286.8	165.6	226.2	17.08	9.86	13.50	5.1
	Day 11	186.2	N/A	186.2	6.62	N/A	6.60	N/A
SD:	Standard deviation

 

Applicant's summary and conclusion

Conclusions:

The test substance was evaluated in an in vitro eye irritation test based on the OECD testing guideline 492 with the Fraunhofer-inhouse developed reconstructed cornea model ( RCE).

Viability was assessed by an MTT assay and barrier function by transepithelial electrical
resistance at 1000Hz (TEER 1000Hz). The viability test shows an irritative effect of the testing substance, suggesting a category 1 or 2 substance.
The TEER 1000Hz indicates a persistent irritative effect over time, indicating a category 1
substance (serious eye damage).

Executive summary:

The test item was evaluated in an in vitro eye irritation test based on the OECD test guideline 492 with the Fraunhofer inhouse developed reconstructed cornea model (RCE).
• Viability was assessed by an MTT assay and barrier function by transepithelial electrical resistance at 1000Hz (TEER 1000Hz).
• the test item was tested as solid substance and was incubated for 6 hours after topical application. Tissue models treated with water (CAS RN: 7732-18-5) were used as negative control or 10 % Benzalkonium chloride (CAS RN: 63449-41-2) as positive control Eight models were used in total
• Internal Acceptance criteria of NC for MTT (2.5>NC>0.8) and TEER (4000>NC>600 ) measurement were met
• the test item revealed a reduction of viability below 60%. As the measured viability was 1% after application of the test item, indicating a category 1 or 2 substance for eye irritation by viability.
• the test item demonstrated a decrease in TEER 1000Hz below 60% with 50% after application that persisted at day 7 with a TEER 1000Hz below 50 % with 14%, indicating a category 1 substance by TEER measurement.